A novel family of defensin-like peptides from Hermetia illucens with antibacterial properties

Abstract

Background: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections.

Results: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa.

Conclusions: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa.

Keywords: Hermetia illucens; Pseudomonas aeruginosa; AlphaFold; Antibiotics; Antimicrobial peptides; Black soldier fly larvae; Defensins.

Fig. 1

Structure predictions of the predicted AMPs; Jg7197.t1, Jg7902.t1, and Jg7904.t1. (A) The tertiary structures of the 3 proteins were predicted using AF2 and the structures were visualised using PyMol. Helical structures are coloured blue; looped regions are coloured magenta and sheets are coloured in red. The 3 proteins held an αβ-structural motif. (B) Superimposition of the 3 AF2 models demonstrated the high degree of similarity between the protein structures. Jg7197.t1 is shown in peach, Jg7902.t1 is shown in light blue and Jg7904.t1 is shown in purple

Fig. 2

Multi-sequence alignments of paralogous gene cluster protein sequences. (A) Sequence alignments of the protein sequences encoded by the gene jg719.t1 cluster and (B) the jg7902.t1 and jg7904.t1 gene cluster. Sequences are coloured dark blue to show high conservation in residues and light blue to show low conservation. Multisequence alignment was performed with TCoffee and visualised with Jalview

Fig. 3

Superimposition of the predicted tertiary structures of the proteins encoded within the novel putative AMP gene clusters. (A) Predicted tertiary structures of proteins encoded by the jg7197.t1-jg7201.t1 gene cluster, and (B) the jg7901.t1-jg7904.t1 gene cluster. (C) Structures from both sets of gene clusters were also overlain to demonstrate that despite the genomic separation between the 2 gene clusters, the structures were highly conserved. In the jg7197.t1-jg7201.t1 gene cluster; Jg7197.t1 is yellow, Jg7198.t1 is light green, Jg7199.t1 is dark blue, Jg7200.t1 is red and Jg7201.t1 is grey. In the jg7901.t1-jg7904.t1 gene cluster; Jg7901.t1 is pink, Jg7902.t1 is light blue, Jg7903.t1 is dark green and Jg790.t14 is purple

Fig. 4

Heterologous expression of recombinant putative AMPs. Recombinant protein constructs containing N-terminal 6xHis and SUMO tags of (A) Jg7197.t1 (23.5 kDa), (B) Jg7902.t1 (23.5 kDa) and (C) Jg7904.t1 (23.8 kDa) were expressed and purified. The recombinant proteins were detected using SDS-PAGE (i), and the presence of the 6xHis tag was confirmed through western blot (ii). Full length gels and blots are shown in Supplementary Fig. 4

Fig. 5

Antimicrobial activity of recombinant Jg7197.t1, Jg7902.t1 and Jg7904.t1. AUCs of OD measurements of bacterial cultures exposed to 250 µg/ml of (A) Jg7197.t1, (B) Jg7902.t1 and (C) Jg7904.t1. * = indicates when the bacteria exposed to the recombinant protein were reduced compared to the growth control population. □ = indicates when the bacteria exposed to the recombinant protein were reduced compared to the buffer population. † = indicates when the growth control population of bacteria was reduced compared to the bacteria exposed to the buffer. Populations labelled with the combination of * □ were reduced in size due to the direct effect of the recombinant protein. (D) Total number of viable cells were measured as CFUs in P. aeruginosa PaO1 cultures exposed to Jg7904.t1 (500 µg/ml, 21.0µM; purple) for 8 h and compared with undisturbed growth (black), and buffer control (grey) populations. 1 = Staphylococcus aureus ATCC 10,788, 2 = S. aureus LGA251, 3 = S. Typhimurium SL1344, 4 = Salmonella enterica serovar Enteritidis NCTC 13,349, 5 = P. aeruginosa PaO1, 6 = Listeria monocytogenes EGD-e, 7 = Klebsiella pneumoniae 43,816, 8 = Escherichia coli K12, 9 = Bacillus thuringiensis serovar finitimus TBt020, 10 = Bacillus subtilis 168, 11 = Bacillus megaterium QM B1551, 12 = Bacillus cereus ATCC 14,579. Statistics were performed using an unpaired Students’ t-test: ns (not significant), **** p ≤ 0.0001